Alarm communication predates eusociality in termites

Termites (Blattodea: Isoptera) have evolved specialized defensive strategies for colony protection. Alarm communication enables workers to escape threats while soldiers are recruited to the source of disturbance. Here, we study the vibroacoustic and chemical alarm communication in the wood roach Cryptocercus and in 20 termite species including seven of the nine termite families, all life-types, and all feeding and nesting habits. Our multidisciplinary approach shows that vibratory alarm signals represent an ethological synapomorphy of termites and Cryptocercus. In contrast, chemical alarms have evolved independently in several cockroach groups and at least twice in termites. Vibroacoustic alarm signaling patterns are the most complex in Neoisoptera, in which they are often combined with chemical signals. The alarm characters correlate to phylogenetic position, food type and hardness, foraging area size, and nesting habits. Overall, species of Neoisoptera have developed the most sophisticated communication system amongst termites, potentially contributing to their ecological success.

Line 157: could include definitions for terminology used in the introduction here (e.g. vibroacoustic signalling, alarm signalling etc) Fig 4 should be changed as the black and blue colour of the boxplots can be hard to distinguish. All figures also have 2 captions, so double check which one should be included in the final manuscript. Fig 6 is missing an axis label on the x axis (vibroacoustic variables plot). As my experience with phylogenetics is limited, I am unable to comment on this aspect of the paper, however, it would be beneficial if you could include how you created the phylogenies used in Figure 2 and 3. Line 178-179-remove "the" in front of general alarm Line 185-187: not convinced by this statement "locomotion activity increased in many cases"-use explicit numbers here because from figure 1, it looks like changes in locomotion activity were not significant most of the time. Line 189-196: you define what the green fields and arrows mean, however you also need to define what the blue boxes with a x are to make things clear. Line 198-206: very interesting observation of termite defensive behaviours! Figure 2: How was the tree in this figure produced? Line 246: include "taxa" or "genera" after Neoisoptera Line 373-375: need references Conclusions/future directions Whilst the authors have summed up the results of the study well, it would be nice to see more about the implications of this research as well as expanding on the future directions of this field as they are only briefly touched upon.

Methods
The methods are somewhat lacking in detail and clarity, with important aspects missing from the section (i.e. how phylogenetic trees used in figures were constructed; sample sizes for behavioural experiments; how treatments were randomised etc.). Line 446: include permit details Line 454: sample sizes needed for experimental groups Line 456-459: were treatments carried out by the same person each time? Variability in researcher technique when carrying out these treatments may influence results Reviewer #2 (Remarks to the Author): The present paper aimed at the evolution of alarm (vibroacoustic and chemical) communication in termites, comparing 20 termite species and a wood roach, Cryptocercus. The multidisciplinary approach applied in this study was important to explain the multiple characters that may be evolved in the evolution of these termite defensive strategies. The results indicate that vibroacoustic signals are a synapomorphy of termites and Cryptocercus, i.e., there is a shared origin in their most recent common ancestror. On the other hand, the authors suggest that chemical signals evolved at least twice in termite species. Given the complexity of termite defensive behaviors, this research is quite interesting and may be useful for future phylogenetic and ecological investigations. The discussions and conclusion are robust and provide very important information for understanding the evolution of alarm communication in termites. However, I have few comments to be addressed by the authors prior to publication. Among the 20 termite species studied, none includes the soldierless termite group. I think that will be interesting to include at least one Apicotermitinae species in discussion, since these termites have peculiar defensive strategies. In Material and Methods, it is important to be clear which species were studied in behavioral and vibroacoustic experiments.
Line 516 -500,000 interactions (not "iteractions"). I hope that my comments will be useful for the improvement of the manuscript.

Reviewer #1 (Remarks to the Author):
Abstract: Generally well written however could benefit from adding information on the knowledge gaps and why you are conducting this research before going into what you studied (Line 62). Additionally, concluding the paragraph on the implications of this research and framing it in the context of animal weaponry and defence on a broader scale will improve this section. => Unfortunately the abstract must be 150 words or fewer according to the guidelines of the journal and we already reached the limit. Therefore, we cannot add additional information.
Lines 65-66: overly wordy sentence with a lot of jargon, consider rephrasing for clarity => We splitted the sentence into two sentences and we rephrased it slightly. We hope it helped.
Significance statement It is still not clear from this section why you have done this study and why this particular study is significant. Be explicit in why you think this research is important in a broad context. => We improved the text for this two aspects.

Introduction:
The introduction is overly verbose, and full of jargon that has not been defined. Terminology is inconsistent throughout (e.g. what is 'alarm signalling'? And is it the same as 'alarm behaviours' and 'alarm communication?'). => We have significantly changed the Introduction in order to make it easier to follow. It is now explained at the line 101 that it is a defensive strategy and more details about alarm signals are provided in the next sentences. Alarm behavior has been defined in the introduction. Alarm communication may be considered as identical to alarm signaling and this information has been added in the text (the latter focus more on the signs themselves).
Consider sticking to one well-defined term rather than changing throughout. => Done.
Additionally, references are sparse throughout the introduction. Given that alarm communication and vibratory signals are ubiquitous in the animal kingdom, it would be beneficial to begin the introduction by framing it in this context and giving examples, rather than diving straight into alarm communication in social animals. => We improved the beginning of the introduction by discussing about alarm communication in animals, and in particular about alarm calls, common in mammals and birds. Many references have been added as well.
=> We added information about the nasus and the self-sacrifice of workers. However, to avoid to expand the text too much, only the existence of symmetrical and asymmetrical snapping mandibles has been mentioned. We are willing to expand more if requested.
Line 119: define "mechanical signaling" => This term is not important and appeared only once so we removed it.
Line 119-122: very long sentence. Consider splitting into 2 separate sentences => We split the sentence into two.
Line 123: remove "the" at start of sentence => Done.
Line 130: remove "the" at start of sentence => Done.
Line 136: "direct observations"-vague. More detail needed => This term has been explained by adding that "In Nature, it is common to see individuals of some social insects warning their nestmates of potential danger." Line 137-140: long-winded sentence-consider splitting into 2 or rephrasing => We split the sentence into two.
Line 157: could include definitions for terminology used in the introduction here (e.g. vibroacoustic signalling, alarm signalling etc) => We used these terms before the terminology section, and so we defined such special terms at the first appearance in the text. => No, it is not. Both plots are the same including the labels, however, in the left one are emphasized the vibroacoustic characters (in blue) while in the right one the ecological ones (in green). We stressed this fact out in the figure heading.

Results and Discussion
As my experience with phylogenetics is limited, I am unable to comment on this aspect of the paper, however, it would be beneficial if you could include how you created the phylogenies used in Figure 2 and 3. => The phylograms are simplified from published phylogenies (refs 83, 109) as stated at line 534.
Line 185-187: not convinced by this statement "locomotion activity increased in many cases"use explicit numbers here because from figure 1, it looks like changes in locomotion activity were not significant most of the time. => The locomotion activity increased in many cases but not most of the time, indeed. This increasing is clearly showed by the green arrows in figure 1 so we do not consider needed to add numbers to explicit the sentence. However, we added that this increasing is particularly obvious in most of the disturbances used in Reticulitermes.
Line 189-196: you define what the green fields and arrows mean, however you also need to define what the blue boxes with a x are to make things clear. => The blue fields with a cross indicate no significant difference. This information has been added.
Line 198-206: very interesting observation of termite defensive behaviours! => Thank you, we believe it too! Figure 2: How was the tree in this figure produced? => General comparison of the evolutionary history of vibroacoustic and chemical alarm distribution among termites and the wood roach Cryptocercus. Topography based on published phylogenies (83, 109). Ancestral states counted according to Maximum Parsimony model. Dots are yellow when the alarm mode is present, white when absent, and grey when unknown. The family name colors represent ranks for MCA analysis.